Hepatocyte spheroids exhibit enhanced liver-specific function compared to hepatocytes cultured as a monolayer. Ultrastructural analysis shows that they also appear to form bile cananlicular-like structures, indicating a more tissue-like structure. Liver-specific cytochrome P450 activity, measured in situ using confocal microscopy, increases over the course of spheroid self-assembly and decreases as cells disassemble into a monolayer. Examination of the diffusion of a fluorescent tracer confirms the formation of a ductal network within spheroids. In order to better demonstrate the formation of differentiated structure, more specific structural markers are being investigated. Preliminary studies have begun to examine the distribution of cytoskeleton and adhesion receptors over the course of spheroid assembly. The ultimate goals are to understand how hepatocytes recognize each other to begin to form spheroids and when the transition to enhanced activity occurs in orda to better exploit hepatocytes for use in tissue engineering applications, such as a bioartificial liver device. We will use the multiphoton imaging system at the IMR to study spheroids labeled by immunofluorescence. We anticipate that the deep-sectioning capabilities of the multiphoton system should allow us to see structures within the spheroids that are inaccessible by conventional confocal microscopy techniques.